PROJECT 3 ABSTRACT/RESEARCH SUMMARY. A high proportion (~70%) of DGAP subjects display neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD), intellectual disability (ID) and others, so DGAP's strategy of identifying the gene(s) disrupted by balanced chromosome abnormalities (BCAs) will continue to be a major source, complementary to other approaches, of human genes whose heterozygous inactivation contributes strongly to NDD. Many NDD genes identified in DGAP function in chromatin regulation and contribute to multiple NDD phenotypes, indicating shared pathways between ASD, ID and adult behavioral disorders. Strong effect mutations in different genes can also lead to a similar phenotype, suggesting the triggering of shared neurodevelopmental pathways. These facts suggest that an unbiased, data-driven genetic strategy using global molecular read-outs will identify shared perturbations in gene expression networks as proximal effects of inactivating different NDD genes. Often the same DGAP subjects also display non-neuronal phenotypes, indicating potential overlaps between developmental pathways in different organ systems, many of which will be the focus of Project 2. Using genes identified in Project 1 from subjects ascertained in the Coordinating and Administrative Core (CAC), Project 3 will test the hypothesis of convergent NDD pathways by creating targeted heterozygous mutations (in close coordination with Projects 1 and 2, especially for cases with both neurological and peripheral abnormalities) and comparing their effects on networks of gene expression in isogenic human induced pluripotent stem cells (iPSC) and differentiating neurons. Networks disrupted in NDDs will allow grouping of different mutated genes (and the subjects who harbor them) based upon their shared consequences and permit efficient deployment by the research community of the genetic and cellular resources of DGAP by targeting the pathways most likely to yield broadly applicable therapeutic interventions. We will further test a basic hypothesis germane to all DGAP cases, that BCAs and truncating mutations limited to the breakpoint-disrupted gene are not identical due to additional effects on gene regulation at a distance (in cis or trans) from changes in the nuclear architecture occasioned by the BCA. Finally, for selected genes, in close coordination with Projects 1 and 2, we will compare genotype-phenotype relationships in human subjects and zebrafish models to cellular phenotypes, both molecular and functional, to provide a firm basis for future studies of detailed mechanisms and possible interventions. Overall, our unbiased, data-driven, genetic strategy will identify alterations of gene networks due to mutations in different genes, potentially revealing critical pathways whose dysregulation leads to NDD and allowing classification of subjects based upon common shared mechanisms. The isogenic mutant human iPSCs (individual gene knock-out and non-truncating allelic series) that we provide for the research community will facilitate further investigation of these mechanisms and development and testing of rational pharmaceutical interventions.